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Although vital pulp therapy should be performed by promoting the wound-healing capacity of dental pulp, existing pulp-capping materials were not developed with a focus on the pulpal repair process. In previous investigations of wound healing in dental pulp, we found that organic dentin matrix components (DMCs) were degraded by matrix metalloproteinase-20, and DMC degradation products containing protein S100A7 (S100A7) and protein S100A8 (S100A8) promoted the pulpal wound-healing process. However, the direct use of recombinant proteins as pulp-capping materials may cause clinical problems or lead to high medical costs. Thus, we hypothesized that functional peptides derived from recombinant proteins could solve the problems associated with direct use of such proteins. In this study, we identified functional peptides derived from the protein S100 family and investigated their effects on dental pulp tissue. We first performed amino acid sequence alignments of protein S100 family members from several mammalian sources, then identified candidate peptides. Next, we used a peptide array method that involved human dental pulp stem cells (hDPSCs) to evaluate the mineralization-inducing ability of each peptide. Our results supported the selection of 4 candidate functional peptides derived from proteins S100A8 and S100A9. Direct pulp-capping experiments in a rat model demonstrated that 1 S100A8-derived peptide induced greater tertiary dentin formation compared with the other peptides. To investigate the mechanism underlying this induction effect, we performed liquid chromatography–tandem mass spectrometry analysis using hDPSCs and the S100A8-derived peptide; the results suggested that this peptide promotes tertiary dentin formation by inhibiting inflammatory responses. In addition, this peptide was located in a hairpin region on the surface of S100A8 and could function by direct interaction with other molecules. In summary, this study demonstrated that a S100A8-derived functional peptide promoted wound healing in dental pulp; our findings provide insights for the development of next-generation biological vital pulp therapies.

Aortic aneurysm (AA) is a vascular disorder characterized pathologically by inflammatory cell invasion and extracellular matrix (ECM) degradation. It is known that regulation of the balance between pro-inflammatory M1 macrophages (M1Ms) and anti-inflammatory M2 macrophages (M2Ms) play a pivotal role in AA stabilization. We investigated the effects of M2M administration in an apolipoprotein E-deficient (apoE-/-) mouse model in which AA was induced by angiotensin Ⅱ (ATⅡ) infusion. Mice received intraperitoneal administration of 1 million M2Ms 4 weeks after ATII infusion. The M2Ms group exhibited reduced AA expansion, decreased expression levels of interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and monocyte chemoattractant protein−1 (MCP)-1, decreased matrix metalloproteinase (MMP)-9 enzymatic activity, and a lower M1M/M2M ratio. Moreover, the M2M group exhibited upregulation of anti-inflammatory factors, including IL-4 and IL-10. PKH26-labeled M2Ms accounted for 6.5% of cells in the aneurysmal site and co-expressed CD206. Taken together, intraperitoneal administration of M2Ms inhibited AA expansion by reducing the inflammatory reaction via M2M dominance. This study shows that M2M administration might be useful for the treatment of AA.

For decades, researchers have investigated the ideal material for clinical use in the cardiovascular field. Several substitute materials are used clinically, but each has drawbacks. Recently we developed biodegradable and elastic poly(ε-caprolactone-co-D,L-lactide) (P(CL-DLLA)) copolymers by adjusting the CL/DLLA composition, and evaluated the long-term efficacy and outcomes of these copolymers when used for right ventricular outflow tract (RVOT) replacement. This P(CL-DLLA) material was processed into a circular patch and used to replace a surgical defect in the RVOT of adult rats. Control rats were implanted with expanded polytetrafluoroethylene (ePTFE). Histologic evaluation was performed at 8, 24, and 48 weeks post-surgery. All animals survived the surgery with no aneurysm formation or thrombus. In all periods, ePTFE demonstrated fibrous tissue. In contrast, at 8 weeks P(CL-DLLA) showed infiltration of macrophages and fibroblast-like cells into the remaining material. At 24 weeks, P(CL-DLLA) was absorbed completely, and muscle-like tissue was present with positive staining for α-sarcomeric actinin and cardiac troponin T (cTnT). At 48 weeks, the cTnT-positive area had increased. The biodegradable and elastic P(CL-DLLA) induced cardiac regeneration throughout the 48-week study period. Future application of this material as a cardiovascular scaffold seems promising. STATEMENT OF SIGNIFICANCE: Biomaterials for reconstruction of tissue deficiencies in cardiovascular surgery require having suitable mechanical properties for cardiac tissue and biodegradation resulting in native tissue growth. Several biodegradable polymers such as poly-ε-caprolactone (PCL) and polylactic acid (PLA) have excellent biocompatibility and already been widely used clinically. In general, PCL and PLA are quite mechanically rigid. Meanwhile, significant elasticity is required in the high-pressure environment of the heart while the material is being replaced by new tissue. The present study provides a novel four-armed crosslinked poly(ε-caprolactone-co-D,L-lactide) (i.e., P(CL-DLLA)) material for cardiac patch, which was demonstrated properties including tissue-compatible, super-elastic nature, that made it suitable for long-term, in vivo RVOT repair. This super-elastic biomaterial could be useful for reconstruction of various muscular tissues deficiencies.

We previously reported the effectiveness of autologous mesenchymal stromal cells (MSCs) for the treatment of aortic aneurysm (AA), mediated mainly by these cells’ anti-inflammatory properties. In this study, we investigate whether the therapeutic effects of allogeneic MSCs on AA are the same as those of autologous MSCs. To examine the immune response to allogeneic MSCs, C57BL/6 lymphocytes were co-cultured with BALB/c MSCs for 5 days in vitro. Apolipoprotein E-deficient C57BL/6 mice with AA induced by angiotensin II were randomly divided into three groups defined by the following intravenous injections: (i) 0.2 ml of saline (n = 10, group S) as a control, (ii) 1 × 106 autologous MSCs (isolated from C57BL/6, n = 10, group Au) and (iii) 1 × 106 allogeneic MSCs (isolated from BALB/c, n = 10, group Al). Two weeks after injection, aortic diameters were measured, along with enzymatic activities of MMP-2 and MMP-9 and cytokine concentrations in AAs. Neither allogenic (BALB/c) MSCs nor autologous (C57BL/6) MSCs accelerated the proliferation of lymphocytes obtained from C57BL/6. Compared with group S, groups Au and Al had significantly shorter aortic diameters (group S vs Au vs Al; 2.29 vs 1.40 vs 1.36 mm, respectively, p

Supplemental material, sj-docx-1-jdr-10.1177_00220345221135766 for Novel Functional Peptide for Next-Generation Vital Pulp Therapy by M. Watanabe, M. Okamoto, S. Komichi, H. Huang, S. Matsumoto, K. Moriyama, J. Ohshima, S. Abe, M. Morita, M. Ali, K. Takebe, I. Kozaki, A. Fujimoto, K. Kanie, R. Kato, K. Uto, M. Ebara, A. Yamawaki-Ogata, Y. Narita, Y. Takahashi and M. Hayashi in Journal of Dental Research

During wound regeneration, both cell adhesion and adhesion-inhibitory functions must be controlled in parallel. We developed a membrane with dual surfaces by merging the properties of carboxymethyl cellulose (CMC) and collagen using vitrification. A rigid membrane was formed by vitrification of a bi-layered CMC and collagen hydrogel without using cross-linking reagents, thus providing dual functions, strong cell adhesion-inhibition with the CMC layer, and cell adhesion with the collagen layer. We referred to this bi-layered CMC-collagen vitrigel membrane as "Bi-C-CVM" and optimized the process and materials. The introduction of the CMC layer conferred a "tough but stably wet" property to Bi-C-CVM. This enables Bi-C-CVM to cover wet tissue and make the membrane non-detachable while preventing tissue adhesion on the other side. The bi-layered vitrification procedure can expand the customizability of collagen vitrigel devices for wider medical applications.

Background. The pathogenesis of abdominal aortic aneurysm (AAA) is characterized by atherosclerosis with chronic inflammation in the aortic wall. Montelukast is a selective cys-LT 1 receptor antagonist that can suppress atherosclerotic diseases. We evaluated the in vitro properties of montelukast and its in vivo activities in an angiotensin II–infused apolipoprotein E–deficient (apoE−/−) AAA mouse model. Methods. The mouse monocyte/macrophage cell line J774A.1 was used in vitro. M1 macrophages were treated with montelukast, and gene expressions of inflammatory cytokines were measured. Macrophages were cultured with montelukast, then gene expressions of arginase-1 and IL (interleukin)-10 were assessed by quantitative polymerase chain reaction, arginase-1 was measured by fluorescence-activated cell sorting, and IL-10 concentration was analyzed by enzyme-linked immunosorbent assay. In vivo, one group (Mont, n=7) received oral montelukast (10 mg/kg/day) for 28 days, and the other group (Saline, n=7) was given normal Saline as a control for the same period. Aortic diameters, activities of matrix metalloproteinases (MMPs), cytokine concentrations, and the number of M2 macrophages were analyzed. Results. Relative to control, montelukast significantly suppressed gene expressions of MMP-2, MMP-9, and IL-1β, induced gene expressions of arginase-1 and IL-10, enhanced the expression of the arginase-1 cell surface protein, and increased the protein concentration of IL-10. In vivo, montelukast significantly decreased aortic expansion (Saline vs Mont; 2.44 ± 0.15 mm vs 1.59 ± 0.20 mm, PμM vs 755 μM, P Conclusion. Montelukast induces M2 macrophage polarization and prevents AAA formation in apoE−/− mice.

Background The cause of enlarged aortic dissection (AD) is associated with increased inflammatory cytokines such as IL-6. Clarithromycin (CAM) has been reported to inhibit inflammatory cytokines via suppressing NF-κB. we investigated whether or not CAM could prevent the enlargement of dissected aortic aneurysm. Methods Male wild type mice (12 weeks of age) were infused with Angiotensin II and 3-aminopropinonitrile for two weeks for AD development. After two weeks, CAM (10 mg/kg/day) or saline was administered orally to the mice every day (CAM group: n=10, SAL group: n=10) with infusing Angiotensin II for more two weeks. After four weeks, the aortic diameter, macrophage infiltration, collagen and elastin quantities, and levels of inflammation related cytokines, were assessed. Results The aortic diameter was significantly suppressed in the CAM group (P < 0.01). No rupture death was observed in the CAM group in contrast to 3 (30%) in the SAL group (P = 0.07). Clarithromycin significantly increased the infiltration of anti-inflammatory macrophages (20.8% vs. 2.8%, P < 0.05). Compared with the controls, the levels of IL-1β (332 pg/mL vs. 800 pg/mL, P < 0.01) and IL-6 (344 pg/mL vs. 727 pg/mL: p < 0.05) were significantly decreased, and the levels of IL-4 (2519 pg/mL vs. 1397 pg/mL, P < 0.05) and TGF-β (1649 pg/mL vs. 1134 pg/mL, P < 0.05) was significantly increased in the CAM group. The collagen area was increased (10.1% vs. 4.2%, P < 0.05) and expression of α-SMA (9.5% vs. 2.8%, P < 0.05) and α-actinin (16.3% vs. 4.0%, P < 0.01) were increased in the CAM group compared with the SAL group. Conclusions CAM suppressed the progression of dissected aortic aneurysm through the anti-inflammatory and the existence of abundant collagen secreted including α-SMA, α-actinin positive cells.

For decades, researchers have investigated the ideal material for clinical use in the cardiovascular field. Several substitute materials are used clinically, but each has drawbacks. Recently we developed poly(e-caprolactone-co-D,L-lactide) (P(CL-DLLA)) polymers with optimized biodegradation and elasticity by adjusting the CL/DLLA composition, and used these polymers in right ventricular outflow tract (RVOT) replacement to evaluate long-term efficacy and outcomes. This P(CL-DLLA) material was processed into a circular patch and used to replace a surgical defect in the RVOT of adult rats. Control rats were implanted with expanded polytetrafluoroethylene (ePTFE). Histologic evaluation was performed at 8, 24, and 48 weeks post-surgery. All animals survived the surgery with no aneurysm formation or thrombus. In all periods, ePTFE demonstrated fibrous tissue. In contrast, at 8 weeks P(CL-DLLA) showed infiltration of macrophages and fibroblast-like cells into the remaining material. At 24 weeks, P(CL-DLLA) was absorbed completely, and muscle-like tissue was present with positive staining for α-sarcomeric actinin and cTnT. At 48 weeks, the cTnT-positive area had increased. The P(CL-DLLA) with optimized elasticity and biodegradation induced cardiac regeneration throughout the 48-week study period. Future application of this material as a cardiovascular scaffold seems promising.

Objective The pathogenesis of aortic aneurysm (AA) is characterized by the chronic inflammation of the aortic wall with the accumulation of macrophages and the degradation of the extracellular matrix (ECM) including elastin. Colchicine (COL) is an alkaloid derived from the plant Lily family Colchicum autumnale, and it is known for anti-inflammatory effects. Plant extracts containing COL have been used in the treatment of gout from ancient period. Currently, pseudogout, familial Mediterranean fever, Behçet's disease and pericarditis are also treated by COL. Furthermore, recent evidence suggests the use of COL for secondary prevention of cardiovascular disease, and the phase 3 clinical trial for it has begun. The objective of this study is to investigate whether COL could prevent the progression of aortic aneurysms. Methods In vitro: Macrophages (J774A.1 cell line) stimulated TNF-α 24 hours before and smooth muscle cell (SMC) were cultured with 10 ng/mL COL, and the gene expression of inflammatory cytokines involved in the AA formation was measured 24 hours later. In vivo: Male apolipoprotein E-deficient mice (30–35 weeks of age) were infused with angiotensin II for 28 days. COL (20 μg/kg/d) or saline (NS, as a control) was administered orally to the mice every day (COL group, n=8; NS group, n=8). Aortic diameter was measured by echography every week and all mice were sacrificed and their thoracoabdominal aorta was harvested at the last day of the administration period and elastin content, MMP activitis, and levels of inflammatory cytokines involved in the AA formation were measured. Results In vitro: The gene expression of IL-1β, TNF-α, MCP-1, NF-κB, MMP-9 in the macrophages was significantly decreased in the COL group. The gene expression of Lox, TIMP-2 in the SMC were significantly increased in COL group. In vivo: Aortic diameter measured by echography every week was significantly suppressed in the COL group (2.25 vs 2.81 mm, p Discussion In AA model mice, COL seems to suppress the progression of AA by anti-infammation and preservation of the ECM structure through the inhibition of NLRP3 inflammasome. That NLRP3 inflammasome activation leads to the progression of AA in AA model mice was previously reported and this supports out results. Methods and Results (in vivo) Conclusions This results suggest that the oral administration of COL prevents the progression of AA in AA model mice and it is expected as a novel therapeutic agent for AA. Acknowledgement/Funding JSPS KAKENHI Grant

Background aims We have confirmed that aortic aneurysm (AA) can be regressed by the administration of bone marrow–derived mesenchymal stromal cells (BM-MSCs). We investigated the kinetics of signaling pathways in AA following treatment with BM-MSCs. Methods Angiotensin II-infused apolipoprotein E–deficient mice were treated by intravenous injection of 1 × 10 6 BM-MSCs in 0.2 mL saline (BM-MSCs group, n = 5) or 0.2 mL saline (saline group, n = 5). Mice were sacrificed 2 weeks after injection and subjected to measurements of the incidence of AA and levels of phosphorylated proteins. Levels of proteins in conditioned media of BM-MSCs were also measured. Results The incidence of AA in the BM-MSCs group was reduced (BM-MSC 40% versus saline 100%, P 0.05). Levels of pNF-kB and pSTAT1 were reduced (pNF-kB: 0.28 versus 0.45 unit/mL, P 0.05, pSTAT1: 0.16 versus 0.34, P 0.05), whereas levels of pAkt and pSmad3 were elevated (pAkt: 0.13 versus 0.07, P 0.01, pSmad3: 1.07 versus 0.47, P 0.05) in the BM-MSCs group. The levels of pNF-kB, pAkt, and pSmad3 were correlated with aortic diameters. Trophic factors including IGFPB-3, NRF, Activin A and PDGF-AA were secreted from BM-MSCs (IGFBP-3: 35.2 pg/mL, NRF: 3.1 pg/mL, Activin A: 3.1 pg/mL, PDGF-AA: 0.45 pg/mL). Conclusions Our findings suggested that the therapeutic mechanism of BM-MSC–mediated AA regression could contribute to regulation of the NF-kB, Smad3 and Akt signaling pathways. In addition, paracrine actions by factors including NRF, IGFBP-3, Activin A and PDGF-AA might have affected these signaling pathways.

Bronchiolitis obliterans syndrome arising from chronic airway inflammation is a leading cause of death following lung transplantation. Several studies have suggested that inhaled hydrogen can protect lung grafts from ischemia-reperfusion injury via anti-inflammatory and -oxidative mechanisms. We investigated whether molecular hydrogen-saturated water can preserve lung allograft function in a heterotopic tracheal allograft mouse model of obliterative airway disease METHODS: Obliterative airway disease was induced by heterotopically transplanting tracheal allografts from BALB/c donor mice into C57BL/6 recipient mice, which were subsequently administered hydrogen water (10 ppm) or tap water (control group) (n = 6 each) daily without any immunosuppressive treatment. Histological and immunohistochemical analyses were performed on days 7, 14, and 21.Hydrogen water decreased airway occlusion on day 14. No significant histological differences were observed on days 7 or 21. The cluster of differentiation 4/cluster of differentiation 3 ratio in tracheal allografts on day 14 was higher in the hydrogen water group than in control mice. Enzyme-linked immunosorbent assay performed on day 7 revealed that hydrogen water reduced the level of the pro-inflammatory cytokine interleukin-6 and increased that of forkhead box P3 transcription factor, suggesting an enhancement of regulatory T cell activity.Hydrogen water suppressed the development of mid-term obliterative airway disease in a mouse tracheal allograft model via anti-oxidant and -inflammatory mechanisms and through the activation of Tregs. Thus, hydrogen water is a potential treatment strategy for BOS that can improve the outcome of lung transplant patients.